RNA viruses include important human pathogens such as hepatitis C, West Nile and dengue viruses and exhibit distinct genetic and immunological properties from DNA viruses. In plants and invertebrates, replication of RNA viruses triggers Dicer-dependent biogenesis of virus-derived small interfering RNAs (siRNAs) to guide specific antiviral immunity by RNA interference (RNAi). However, direct evidence supporting a similar antiviral role for the siRNA pathway in mammals is not available. We have obtained key mechanistic insights to the RNA-based antiviral immunity using Flock house virus infection of the fruit fly Drosophila melanogaster, which has been a powerful model for studies on both RNAi and innate immunity. We have recently reported production of a novel class of virus-derived small RNAs in cultured Drosophila cells known as PIWI- interacting RNAs (piRNAs), which are produced independently of Dicer in flies and mammals to silence transposable elements in the germline. Further genetic studies indicate a novel antiviral function for the piRNA pathway in the fruit fly that inhibits virus vertical transmission to the progeny. Ai 1 will determine the genetic requirements of the piRNA-directed antiviral defense and investigate the population, biogenesis and activity of viral piRNAs. Aim 2 chose an arthropod-borne mammalian RNA virus that encodes a viral suppressor of RNAi (VSR) to examine the hypothesis that antiviral RNAi is induced in the mammalian cells during the early stages of viral RNA replication. We found that infection of a VSR-deficient mutant of the virus was effectively suppressed in mammalian cells and was associated with production of viral siRNAs that share key properties with the viral siRNAs produced in the fruit fly in response to Flock house virus infection. Aim 2 will investigate the biogenesis of viral siRNAs in mammalian cells and determine if the detected viral siRNAs direct specific antiviral defense in the mammalian host cells. These studies will likely open up a new area of research on the function of viral piRNAs and establish a mammalian system to investigate the biogenesis and antiviral function of virus-derived small RNAs.